1. Technical Field
The subject invention relates to a method in which the polymerase chain reaction is used to add homologous ends to DNA. These homologous ends undergo recombination in vivo following transfection of E. coli with linear PCR products. Therefore, the placement of homologous ends by PCR permits the formation of DNA joints in vivo. If the recombinant construct contains plasmid sequences that permit replication and a selectable phenotype, E. coli can be transformed with the construct of interest. Thus, the method can be used for the rapid generation of recombinant DNA constructs and for the generation of site-specific mutants.
2. Background Information
Two underpinnings of the recombinant DNA revolution are the capacity to recombine DNA, and the capacity to mutate a specific site in a DNA segment. Recently, the polymerase chain reaction (PCR) has gained use as a rapid means for the site-specific mutagenesis of DNA and for the recombination of DNA.
The polymerase chain reaction (PCR) is a method by which a specific DNA sequence can be amplified in vitro (U.S. Pat. Nos. 4,683,202, 4,683,195, Mullis et al., Cold Spring Harbor Symposia on Quantitative Biology, Vol. LI: 263-73 (1986), Saiki et al., Science 230:1350-54 (1985)). Prior investigators have used PCR to generate site-specific mutants (Hemsley et al., Nucleic Acids Res. 17:6545-51 (1989), Higuchi et al., Nucleic Acids Res. 16:7535-67 (1988), Ho et al., Gene 77:51-59 (1989), Kadowaki, Gene 76:161-66 (1989), Kammann et al., Nucleic Acids Res. 17:5404 (1989), Nelson et al., Anal. Biochem. 180:147-51 (1989) and Vallette et al., Nucleic Acids Res. 17:723-33 (1989)). PCR has also been used to amplify inserts which later undergo a separate subcloning procedure (Saiki et al., Science 239:487-91 (1988)).
Site-specific mutants are created by introducing mismatches into the oligonucleotides used to prime the PCR amplification. These oligonucleotides, with their mutant sequence, are incorporated into the PCR product. PCR has also been used to join segments of DNA by a method called splicing by overlap extension (Higuchi et al., Nucleic Acids Res. 16:7351-67 (1988), Ho et al., Gene 77:51-59 (1989), Horton et al., Gene 77:61-68 (1989)). This requires two sequential PCR amplifications, and is not designed to generate cohesive ends. Therefore, cloning of the product requires additional in vitro enzymatic manipulations following the PCR amplification.
A recent report describes a method for site-specific mutagenesis based on amplification of the entire plasmid (Hemsley et al., Nucleic Acids Res.. 17:6545-51 (1989)). In that protocol, the ends of the PCR product are treated with the Klenow fragment of DNA polymerase 1. Subsequently, these ends are phosphorylated at the 5' terminus with Polynucleotide Kinase prior to an in vitro self-annealing blunt end ligation reaction.
The present inventor recently discovered a method for site-specific mutagenesis and for DNA recombination that requires no enzymatic reaction in vitro apart from DNA amplification (U.S. application Ser. No. 07/432,993 (filed on Nov. 8, 1989), Jones et al., BioTechniques, 8:178-83 (1990) and Jones et al., Nature 344:793-94 (April 1990)). This is accomplished by using separate PCR amplifications to generate products that when combined, denatured and reannealed form double-stranded DNA with discrete cohesive single-stranded ends that are complementary to each other and anneal to form DNA circles suitable for transformation of E. coli. This approach is termed recombinant circle PCR (RCPCR).
Others have used the ability of E. coli to recombine short stretches of homology to generate site-specific mutants in a method called the crossover linker technique. The crossover linker technique generates homologous ends, and the mutation of interest, by ligating a synthetic linker to a restriction enzyme digested plasmid (Sung et al., Gene 47:261-67 (1986) and Sung et al., DNA, 6:373-79 (1987)). This method can readily be distinguished from that of the subject invention.
All U.S. patents and publications referred to herein are hereby incorporated by reference.